Synaptic vesicle (SV) exocytosis is mediated by SNARE proteins that form the core of a membrane fusion machine; this machine is precisely controlled by regulatory proteins. Rapid, synchronous Ca2+-triggered SV fusion is triggered by the tandem C2-domain Ca2+-binding protein synaptotagmin 1 (syt1), but the mechanism of action of syt1 remains unclear. In Aim 1, we address this issue three ways. In the first sub-Aim, we will exploit a new and powerful assay that we developed in which SNARE-bearing nanodiscs dock and fuse with cognate SNARE-bearing black lipid bilayers, thus enabling the first electrophysiological recordings of entirely recombinant fusion pores. This system affords sec time resolution to reveal fundamental properties of individual fusion pores. This advance makes it possible to directly address the impact of wt and mutant forms of syt1 on the conductance and kinetic properties of pores. We will test the idea that different structural elements of syt1 regulate pore opening versus dilation. In the second sub-Aim, we will build on our new preliminary finding that the C2B domain (i.e. the second Ca2+ binding motif in the primary sequence), but not the C2A domain (the first Ca2+ binding motif), is both necessary and sufficient to trigger robust exocytosis in neurons. We will graft structural elements of C2B onto C2A to conduct gain-of-function experiments that will reveal which elements, and hence effector interactions, enable C2B to trigger fusion. In the third sub-Aim, we will determine the biochemical and functional deficits of novel syt1 mutations recently identified in two human patients, thus uncovering the molecular basis for the debilitating conditions that result from these mutations. There are seventeen isoforms of syt, and our long term goal is to elucidate the functions of each of them. In Aim 2, we focus on one of the least studied isoforms, syt17 (also called B/K protein). No function has been assigned to syt17, but it is expressed in a number of brain regions and has been associated with autism. Our preliminary studies indicate that syt17 plays a role in neurite outgrowth, synaptic transmission, and behavior. We will study the interaction of this syt isoform with Ca2+ and other molecules, determine its subcellular localization and trafficking, conduct a detailed analysis of its impact on neuronal morphology and neurite outgrowth kinetics, and determine whether it regulates axonal regeneration after injury. Also, because syt17 has been implicated in a neurological disorder, we will continue to study how loss of this protein impacts synaptic transmission and behavior in mice. Together, the two Aims proposed here will provide important new insights into the function of syt1 and syt17, and will highlight how members of this protein family have functionally diverged to regulate vastly different aspects of neuronal cell biology.